Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 366-370
ISSN: 2319-7706 Volume 4 Number 4 (2015) pp. 366-370
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Original Research Article
Comparative study of three phenotypic methods for detection of Metallo- lactamases in clinical isolates of Pseudomonas aeruginosa
Sonia Sharma*, Rama Sikka, Antariksh Deep, Seema Mittal,
Aakanksha Sharma and Uma Chaudhary
Department of Microbiology, Pt. B.D. Sharma, Post Graduate Institute of
Medical Sciences, Rohtak, Haryana, India
*Corresponding author
ABSTRACT
Keywords
Pseudomonas
aeruginosa,
Imipenem,
resistance,
phenotypic
methods
The acquisition of metallo- -lactamases (MBL) by P. aeruginosa has recently emerged as one of the
most worrisome resistance mechanisms, a virtue by which they can hydrolyze all beta-lactam
antibiotics including penicillins, cephalosporins and carbapenems, with the exception of aztreonam.
This study was
undertaken
to
detect
and
compare
production
of metallo- lactamases in clinical isolates of P.aeruginosa by three different phenotypic methods.
This study was conducted over a period of one year (January to December 2012), in the Department
of Microbiology, Pt. B.D. Sharma, Post Graduate Institute of Medical Sciences, Rohtak. A total of
100 clinical isolates were included in the study and their antimicrobial susceptibility was determined
by Kirby-Bauer disc diffusion method. The screening for MBL detection was done by imipenemEDTA combined disc test, imipenem-EDTA double disc synergy test and EDTA disc potentiation test
using four cephalosporins, in the Imipenem resistant isolates. Imipenem resistance was seen in a total
of 65 P. aeruginosa isolates. Screening for MBL was positive in 57 isolates by combined disc test,
56 isolates by double disc synergy test and 54isolates by disc potentiation test. Conclusion:
Imipenem-EDTA combined disc test was superior to other methods for MBL detection in clinical
Microbiology laboratories.
Introduction
(Manoharan, 2010). The MBLs are
associated with a peculiar property of
conferring upon the organism a broadspectrum resistance profile. The presence of
MBLs
is
usually associated
with
accompanying resistance to
-lactams,
aminoglycosides and fluoroquinolones as
well, although remaining sensitive to
polymyxins. Therefore, a rapid screening
method to detect MBLs in multidrug
resistant P. aeruginosa isolates, could help
in modifying therapy and initiating effective
infection control measures to prevent further
dissemination.
Pseudomonas aeruginosa, a commonly
encountered nosocomial pathogen, is a
potentially troublesome cause of wound
infection, urinary tract infection, and
respiratory tract infection, especially in
immunocompromised
patients,
thus,
inflicting
significant
morbidity
and
mortality, worldwide (Khan, 2008).
Emergence of resistance to commonly used
antibiotics like beta-lactams has been
recognized as a cause of treatment failure
(Carmelli, 1999). In P. aeruginosa,
resistance to carbapenems mediated by
production of metallo beta lactamases
(MBLs) is being increasingly reported
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Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 366-370
Presently, no one standardized phenotypic
method for MBL detection has been
proposed. Despite polymerase chain reaction
being a highly accurate and reliable method,
its accessibility is often limited to reference
laboratories. The present study was thus
conducted with an objective to detect the
prevalence of MBLs in P. aeruginosa
isolates by three different phenotypic
methods and compare their relative efficacy.
units), colistin (10µg). In case of urinary
isolates, ofloxacin (5µg) and norfloxacin
(10µg) were also included. P. aeruginosa
ATCC 27853 strain was employed as the
control strain (Winn Jr et al, 2006;Sussman,
1998).
An isolate was considered as multi drug
resistant (MDR), if resistance was
encountered to least three of the following
classes of antimicrobial agents, i.e lactams, carbapenems, aminoglycosides, and
fluoroquinolones; and, extremely drug
resistant if found to be resistant to all the
four classes of antimicrobial agents
mentioned previously.
Material and Methods
This study was conducted over a period of
one year (January to December 2012), in the
Department of Microbiology, Pt. B.D.
Sharma, Post Graduate Institute of Medical
Sciences, Rohtak, a tertiary level health care
providing facility in North India.
The protocol for the MBL detection in the P.
aeruginosa isolates by the three methods
used in the study is given as under:
A total of 100 isolates of P. aeruginosa
recovered from clinical specimens like
urine, pus, blood, body fluids, sputum, etc
were included in the study. The clinical
isolates recovered from both outdoor and
indoor patients, irrespective of their age and
gender, were identified following standard
microbiological
procedures
(Collee,
1996a,b).
Imipenem (IMP)-EDTA combined disc
test
The test organism was inoculated on MHA
plate as per CLSI guidelines. Two 10µg
imipenem discs were placed on the surface
of agar plate and EDTA solution was added
to one disc to obtain a desired concentration
of 750µg. The inhibition zone of imipenem
and imipenem-EDTA disc was compared
after 16-18 hours of incubation at 35ºC. A
positive test was indicated if zone
enhancement with EDTA impregnated
imipenem discs was 7mm than imipenem
disc alone (Yong et al, 2002).
Antimicrobial Susceptibility
Antimicrobial susceptibility testing was
performed by Kirby-Bauer disc diffusion
method as per CLSI guidelines (CLSI,
2011).
Following antimicrobials discs were tested:ceftazidime (30µg), cefepime (30µg),
ceftriaxone (30µg), ceftizoxime (30µg),
cefoxitin (30µg), imipenem (10µg),
meropenem (10µg), piperacillin/tazobactam
(100/10µg),
ticarcillin/clavulanic
acid
(75/10µg), aztreonam (30µg), gentamicin
(10µg), amikacin (30µg), netilmicin (30µg),
ciprofloxacin (5µg), polymyxin B (300
Imipenem (IMP)-EDTA
synergy test (DDST)
double
disc
Test organism was inoculated on MHA and
a 10µg imipenem disc was placed 20 mm
centre to centre from a blank disc containing
10µl of 0.5M EDTA (750µg). Enhancement
of the zone of inhibition in the area between
imipenem and the EDTA disc in comparison
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Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 366-370
with the zone of inhibition on the far side of
the drug was interpreted as a positive result
(Lee et al, 2003).
standard for detection of MBL, different
studies have reported results using different
methods. In the current study, out of 100
isolates of P. aeruginosa, imipenem
resistance was observed in 65.0% of the
isolates. Out of these isolates, 87.7% were
MBL producers by imipenem (IMP)-EDTA
combined disc test, 86.15% by Imipenem
(IMP)-EDTA(DDST) and 83.07% by EDTA
disc
potentiation
test(Table.1).
The
difference in production rates was however
not statistically significant. Similarly other
studies have also reported 85.7% and 66.7%
detection of MBL by imipenem (IMP)EDTA combined disc test, whereas MBL
detection by Imipenem (IMP)-EDTA
(DDST) has been reported to be 64.28%,
50.0% and by EDTA disc potentiation test
has been reported to be 0.0%, 11.1%
(Behera et al, 2008; Bhalerao et al, 2010).
This suggests that imipenem (IMP)-EDTA
combined disc test is superior to the other
two methods. Silpi et al reported 90.2%
MBL production in imipenem resistant P.
aeruginosa isolates by imipenem (IMP)EDTA combined disc test, imipenem (IMP)EDTA double disc synergy test and MBL Etest strip, thus suggesting all the three
methods to be equally effective in detecting
MBL production (Silpi et al, 2012).
EDTA
disc
potentiation
using
ceftazidime, ceftizoxime, cefepime and
cefotaxime
Test organism was inoculated on to the
Mueller-Hinton agar plates as per CLSI
guidelines and a filter paper blank disc was
placed. Following discs [ceftazidime (30µg),
ceftizoxime (30µg), cefotaxime (30µg),
cefepime (30µg)] were placed 25mm centre
to centre from the blank disc. Ten µl of
0.5M EDTA solution was added to the blank
disc and the plate was incubated overnight at
350C. Enhancement of the zone of inhibition
in the area between the EDTA disc and any
one of the four cephalosporin discs in
comparison with the zone of inhibition on
the far side of the drug was interpreted as a
positive result (Arkawa et al, 2000).
Result and Discussion
Production of MBL by P. aeruginosa and
other gram negative bacteria has tremendous
impact on therapeutic regimens, since these
organisms also carry other multidrug
resistance genes and the only viable
treatment options remains the administration
of potentially toxic polymyxin B and colistin
(Livermore and Woodford, 2000). In the
present study also, 74% of the MBL
producing P. aeruginosa isolates were found
to multi drug resistant (MDR) and 60% were
extremely drug resistant (XDR) isolates.
MBL producing strains are more likely to
cause invasive disease and are associated
with a higher hospital case-fatality rate.
Therefore, it becomes important to have a
rapid screening test to detect MBLs in these
isolates so that further dissemination can be
prevented (Noyal et al, 2009).
The prevalence of MBL detection by
imipenem-EDTA combined disc test has
been reported to range from 8.0%-85.7% in
various studies (Collee et al, 1996; Noyal et
al, 2009; Behera et al, 2008; Livermore,
2002). So, the rate of MBL production was
comparatively higher in the current study.
This can be explained by the fact that
because of increase in ESBL producing
isolates, use of carbapenems to treat serious
infections in our institute has increased and
thus lead to antibiotic selection pressure
under the influence of which organisms
starts
expressing
many
resistance
mechanisms for its survival. This might be
responsible for production of MBL enzymes
Since there is no one test considered as gold
368
Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 366-370
in P. aeruginosa isolates in the present study
(Noyal et al, 2009; Agarwal et al, 2008;
Navneeth et al, 2002). However, another
author reported a higher rate (94.20%) of
MBL production in imipenem resistant P.
aeruginosa isolates by imipenem (IMP)EDTA combined disc test, which can be due
to the fact that study was performed only on
isolates recovered from burn wound samples
(Saderi et al, 2010). Absence of MBL
production in imipenem resistant isolates
may be due to other resistance mechanisms
involved such as permeability mutations via
loss of porins or upregulation of efflux
systems which may be missed by phenotypic
tests (Saderi et al, 2010).
Table.1 Comparison of three methods employed for the detection of MBL
among imipenem resistant P. aeruginosa isolates
Total Number
of imipenem
resistant
P. aeruginosa
isolates out of
100
65
MBL Isolates,
detected by
imipenem (IMP)EDTA combined
disc test
MBL Isolates, detected
by imipenem (IMP) EDTA double disc
synergy test (DDST)
MBL Isolates, detected
by EDTA disc
potentiation method
87.7%
86.15%
83.07%
References
Agarwal G, Lodhi RB, Kamalakar UP,
Khadse RK, Jalgaonkar SV. 2008.
Study of metallo-beta lactamase
production in clinical isolates of
Pseudomonas aeruginosa. Indian J
Med Microbiol., 26: 349-51.
Arkawa Y, Shibata N, Shibayama K,
Kurokawa H, Yogi T, Fusiwara H et
al. 2000. Convenient test for
screening
metallo-beta-lactamases
producing gram-negative bacteria
using thiol compounds. J Clin
Microbiol., 38: 40 -3.
Behera B, Mathur P. Das A, Kapil A,
Sharma V., 2008. An evaluation of
four different phenotypic techniques
for detection of metalo- -lactamase
producing Pseudomonas aeruginosa.
Indian J Med Microbiol., 26: 233-7.
Bhalerao DS, Roushani S. Kinikar AG,
Akhtar I. 2010. Study of MBL
producing P. aeruginosa in pravara
rural hospital. Pravara Med Rev., 5:
16-9.
Carmelli Y, Troillet N, Eliopoulos GM,
Samore MH. 1999. Emergence of
antibiotic resistant Pseudomonas
aeruginosa: comparison of risks
associated
with
different
antipseudomonal agents. Antimicrob
Agents Chemother., 43: 1379-82.
Clinical and Laboratary Standards Institute.
2011. Performance standards for
antibiotic disk susceptibility tests:
approved standard. 9th ed. CLSI
Document M100-S21,Vol 31(1).
Wayne, PA.
Collee JG, Mles RB, Watt B. 1996. Tests for
identification of bacteria. In : Collee
JG, Fraser AG, Marmon BP,
Simmons A, editors. Mackie and
McCartney
Practical
Medical
th
Microbiology, 14
ed. Churchill
Livingstone, New York, Pp131-49.
Collee JG, Duguid JP, Fraser AG, Marmion
BP, Simmons A. 1996. Laboratory
strategy in the diagnosis of infective
syndromes. In : Collee JG, Fraser AG,
369
Int.J.Curr.Microbiol.App.Sci (2015) 4(4): 366-370
Marmion BP, Simmons A, editors
Mackie and McCartney Practical
Medical Microbiology, 14th ed.
Churchill Livingstone, New York, Pp
53-94.
Khan JA, Igbal Z, Rahman SU, Farzana K,
Khan A. 2008. Prevalence and
resistance pattern of Pseudomonas
aeruginosa
against
various
antibiotics. Pak J Pharm Sci., 21:
311-5.
Lee K, Lim YS, Yong D, Yum JH, Chong
Y. 2003. Evaluation of the Hodge test
and the imipenem-EDTA double disk
synergy test for the differentiation of
the metallo- -lactamase producing
clinical isolates of the Pseudomonas
spp and the Acinetobacter spp. J Clin
Microbiol., 41: 4623-9.
Livermore DM, Woodford N. 2000.
Carbapenemases: a problem in
waiting? Curr Opin Microbiol., 3:
489-95.
Livermore
DM.
2002.
Multicentre
evaluation of VITEK 2 Advanced
Expert System for interpretive reading
of antimicrobial resistance tests. J
Antimicro Chemother., 49: 289-300.
Manoharan A, Chatterjee S, Mathai D,
SARI Study Group. 2010. Detection
of metallo- -lactamase producing
Pseudomonas aeruginosa. Indian J
Med Microbiol., 28: 2414.
Navneeth BV, Sridaran D, Sahay D,
Belwadi MA. 2002. Preliminary study
on
the
metallo
betalactamase
producing Pseudomonas aeruginosa
in hospitalised patients. Indian J Med
Res., 112: 264-67.
12.
Saderi H, Lotfalipour H, Owlia P, Salimi H.
2010. Detection of metallo- lactamase producing P. aeruginosa
isolated from burn patients in Tehran,
Iran. LABMEDICINE., 41: 609-11.
Silpi Basak, Ruchita O. Attal and Monali
Rajurkar . 2012. Pseudomonas
Aeruginosa and Newer -Lactamases:
An Emerging Resistance Threat,
Infection Control - Updates, Dr.
Christopher Sudhakar (Ed.), ISBN:
978-953- 51-0055-3, InTech,
Sussman M. 1998. Urinary tract infections.
In: Collier L, Balows A, Sussman M,
editors.
Topley
&
Wilson,s
Microbiology
and
Microbial
infections. 10th ed. Arnold, Great
Britain, Pp601-21.
Winn Jr. WC, Allen SD, Janda WM,
Koneman
EW,
Procop
GW,
Schrekenberger PC, et al editors.
2006. The Non Gram-Negative
Bacilli. In : Koneman's color Atlas
and
Textbook
of
Diagnostic
Microbiology. 6th ed. Lippincott
Williams and Wilkins, Philadelphia,
Pp.946-1014.
Yong D, Lee K, Yum JH, Shin H B,
Rossolinism, Chong Y. 2002.
Imipenem- EDTA disk method for
differentiation
of
metallo-betalactamases producing clinical isolates
of
Pseudomonas
spp
and
Acinetobacter spp. J Clin Microbiol.,
40: 3798 -801.
Noyal MJC, Menezes GA, Harish BN,
Sujatha S, Parija SC. 2009. Simple
screening tests for detection of
carbapenemases in clinical isolates of
non-fermentative
gram
negative
bacteria. Indian J Med Res., 129: 707370